Neuroinflammation is an integral component of neurodegenerative disorders, CNS infection and trauma. Astroglial chemokines, such as CXCL10, are instrumental in neuroinflammatory signaling as well as neurotoxicity. We have utilized proinflammatory-induced CXCL10 expression in normal human astrocytes (NHA) as a model in which to assess the anti-inflammatory actions of the selective, mu-opioid receptor (MOR) antagonist, β-funaltrexamine (β-FNA). Interferon (IFN)γ + HIV-1 Tat-induced CXCL10 expression (secreted protein and mRNA) was inhibited by co-treatment with β-FNA. Neither the MOR-selective antagonist, D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH2 (CTAP) nor the nonselective opioid receptor antagonist, naltrexone inhibited IFNγ + HIV-1 Tat-induced CXCL10 expression. Furthermore, co-treatment with excess CTAP or naltrexone did not prevent β-FNA mediated inhibition of IFNγ + HIV-1 Tat-induced CXCL10 expression. Additionally, we utilized an inhibitor of NF-κB activation (SN50) to demonstrate that IFNγ + HIV-1 Tat-induced CXCL10 expression is NF-κB-dependent in NHA. Subsequent experiments revealed that β-FNA did not significantly affect NF-κB activation. Interestingly, we discovered that β-FNA inhibited p38 activation as indicated by decreased expression of phospho-p38. Together, these findings suggest that the inhibitory actions of β-FNA are MOR-independent and mediated, in part, via a transcriptional mechanism. These findings add to our understanding of the mechanism by which chemokine expression is inhibited by β-FNA. In conjunction with future investigations, these novel findings are expected to provide insights into the development of safe and effective treatments for neuroinflammation.
astroglial; HIV-1; mu opioid receptor; neuroinflammation; interferon γ
Neuroinflammation and neuronal degeneration observed in Parkinson’s disease (PD) has been attributed in part to glial-mediated events. Increased expression of proinflammatory cytokines and abnormal accumulation of the neuronal protein, α-synuclein in the brain are also characteristic of PD. While increasing evidence suggests that astrocytes contribute to neuroinflammation and dopaminergic neuronal degeneration associated with PD, there remains much to learn about these astroglial-mediated events. Therefore, we investigated the in vitro effects of interleukin-1β (IL-1β) and α-synuclein on astroglial expression of interferon-γ inducible protein-10 (CXCL10), a proinflammatory and neurotoxic chemokine. IL-1β-induced CXCL10 protein expression was potentiated by co-exposure to α-synuclein. α-Synuclein did not significantly affect IL-1β-induced CXCL10 mRNA expression, but did mediate increased CXCL10 mRNA stability, which may explain, in part, the increased levels of secreted CXCL10 protein. Future investigations are warranted to more fully define the mechanism by which α-synuclein enhances IL-1β-induced astroglial CXCL10 expression. These findings highlight the importance of α-synuclein in modulating inflammatory events in astroglia. These events may be particularly relevant to the pathology of CNS disorders involving α-synuclein accumulation, including PD and HIV-1 associated dementia.
Parkinson’s disease; alpha-synuclein; astrocyte; neuroinflammation; chemokine; NF-κB
Mercury is neurotoxic and increasing evidence suggests that environmental exposure to mercury may contribute to neuropathologies including Alzheimer's disease and autism spectrum disorders. Mercury is known to disrupt immunocompetence in the periphery, however, little is known about the effects of mercury on neuroimmune signaling. Mercury-induced effects on central immune function are potentially very important given that mercury exposure and neuroinflammation both are implicated in certain neuropathologies (i.e., autism). Furthermore, mounting evidence points to the involvement of glial activation in autism. Therefore, we utilized an in vivo model to assess the effects of mercury exposure on neuroimmune signaling. In prairie voles, 10 week mercury exposure (60 ppm HgCl2 in drinking water) resulted in a male-specific increase in TNFα protein expression in the cerebellum and hippocampus. These findings are consistent with our previously reported male-specific mercury-induced deficits in social behavior and further support a role for heavy metals exposure in neuropathologies such as autism. Subsequent studies should further evaluate the mechanism of action and biological consequences of heavy metals exposure. Additionally, these observations highlight the potential of neuroimmune markers in male voles as biomarkers of environmental mercury toxicity.
heavy metals; environmental toxins; voles; cytokines; chemokines; autism
Under selective pressure from host immunity, viruses have retained genes encoding “immunoevasins” - molecules interfering with host viral recognition and clearance. Due to their binding specificities, immunoevasins can be exploited as affinity labels to identify host-encoded molecules of previously unsuspected importance in defense against the relevant class of virus. We previously described an orthopoxvirus MHC class I-like protein (OMCP) that binds with high affinity to the activating receptor NKG2D on NK and T cell subsets, implicating NKG2D in anti-orthopoxvirus immunity. Here, we report that OMCP also binds in an NKG2D-independent manner to B cells and monocytes/macrophages. We identify murine Fc receptor-like 5 (FCRL5), an orphan immunoregulatory protein highly expressed by innate B lymphocytes, as a specific receptor for OMCP. The three N-terminal Ig domains of FCRL5 are required for OMCP binding. The targeting of FCRL5 by an orthopoxvirus immunoevasin strongly implicates it in contributing to host defense against zoonotic orthopoxviruses.
Fc receptor-like A (FCRLA) is an unusual member of the extended Fc receptor family. FCRLA has homology to receptors for the Fc portion of Ig (FCR) and to other FCRL proteins. However, unlike these other family representatives, which are typically transmembrane receptors with extracellular ligand-binding domains, FCRLA has no predicted transmembrane domain or N-linked glycosylation sites and is an intracellular protein. We show by confocal microscopy and biochemical assays that FCRLA is a soluble resident endoplasmic reticulum (ER) protein, but it does not possess the amino acid sequence KDEL as an ER retention motif in its C-terminus. Using a series of deletion mutants, we found that its ER retention is most likely mediated by the amino terminal partial Ig-like domain. We have identified ER-localized Ig as the FCRLA ligand. FCRLA is unique among the large family of Fc receptors, in that it is capable of associating with multiple Ig isotypes, IgM, IgG and IgA. Among hemopoietic cells, FCRLA expression is restricted to the B lineage and is most abundant in germinal center B lymphocytes. The studies reported here demonstrate that FCRLA is more broadly expressed among human B lineage cells than originally reported; it is found at significant levels in resting blood B cells and at varying levels in all B-cell subsets in tonsil.
B lymphocyte; ER retention; Fc receptor; membrane and secretory Ig; microsomes
Increasing evidence indicates neuroinflammation is instrumental in the pathogenesis of Parkinson's disease (PD). In PD, there is selective degeneration of neuromelanin (NM)-containing dopamine neurons. Neuromelanin is predominantly cytoprotective within dopaminergic neurons, whereas, NM released from damaged neurons activates microglia. However, the effects of NM on astroglial cells remain largely unknown. Astroglia are essential to neuronal homeostasis and responsive to injury, in part, through secretion of chemokines, including interferon γ inducible protein-10 (CXCL10). Thus, we used an in vitro approach to identify the effects of NM on TNFα-induced CXCL10 expression in human astroglial cells. TNFα-induced CXCL10 expression was inhibited in NM exposed cells. Additionally, TNFα-induced NF-κB activation was inhibited by NM. Given that CXCL10 expression is NF-κB-dependent in human astroglial cells, these findings suggest that NM may inhibit CXCL10 expression, in part, through an NF-κB-dependent mechanism. While the in vivo consequences of NM mediated effects on astroglial CXCL10 expression remain to be fully elucidated, insights obtained in this study further our understanding of the effects of NM on inflammatory signaling in human astroglial cells.
Parkinson's disease; chemokine; astrocytes; neuroinflammation; NF-κB
Treatment of SK-N-SH cells with morphine and interleukin-1beta (IL-1β) produced dual regulation of the mRNA for the human mu opioid receptor (MOR) protein. Morphine produced a decrease in the MOR mRNA while IL-1β increased it, as assessed by real-time quantitative PCR. These data were consistent with immunocytochemical studies of treated and untreated cells. Morphine-mediated down-regulation of MOR was blocked by naltrexone and IL-1β-induced up-regulation of MOR was blocked by interleukin-1 receptor type 1 antagonist. Immune-opioid crosstalk was examined by IL-1β and morphine co-treatment. These data are the first to show dual regulation of MOR in neuroblastoma cells.
interleukin-1β; morphine; mu opioid receptor; SK-N-SH cells; opioid-immune crosstalk
The inducible isoform of nitric-oxide synthase (iNOS) is involved in neuropathogenesis associated with infection and disease in the brain. Hence, there is considerable interest in the identification of therapeutic interventions to prevent iNOS-mediated pathology. Astroglia are a major site of iNOS expression during neuropathogenesis. To mimic a key component of neuroinflammation, human A172 astroglial cells were exposed in vitro to a cytokine mixture containing interferon γ, tumor necrosis factor α, and interleukin-1β, resulting in significant iNOS expression. Next, we assessed the effects of the mu opioid receptor antagonist, β-funaltrexamine (β-FNA), on cytokine induced iNOS expression in human astroglia. β-FNA dose-dependently inhibited iNOS expression. β-FNA transcriptionally (or pre-transcriptionally) inhibited cytokine-induced iNOS activation as indicated by a significant decrease in NOS2 messenger RNA expression. Further characterization of the novel, anti-inflammatory actions of β-FNA may provide insights for pharmacologic strategies to treat or prevent brain pathologies associated with neuroinflammation.
neuroinflammation; opioids; astrocytes; nitric oxide; fentanyl; morphine
Given its catalytic activity to generate diacylglycerol and inositol 1,4,5-trisphosphate (IP3), phospholipase C (PLC) is implicated in promoting cell growth. However, we found that PLC-β3-deficient mice develop myeloproliferative disease (MPD), lymphoma, and other tumors. The mutant mice have increased numbers of hematopoietic stem cells (HSC) with increased proliferative, survival, and myeloid-differentiative abilities. These properties are dependent on Stat5 and can be antagonized by the protein phosphatase SHP-1. Stat5-dependent cooperative transformation by active c-Myc and PLC-β3 deficiency was suggested in mouse lymphomas in PLC-β3−/− and in Eμ-myc;PLC-β3+/− mice and human Burkitt's lymphoma cells. The same mechanism for malignant transformation seems to be operative in other human lymphoid and myeloid malignancies. Thus, PLC-β3 is likely a tumor suppressor.
HIV encephalitis (HIVE), the pathologic correlate of HIV-associated dementia (HAD) is characterized by astrogliosis, cytokine/chemokine dysregulation and neuronal degeneration. Increasing evidence suggests that inflammation is actively involved in the pathogenesis of HAD. In fact, the severity of HAD/HIVE correlates more closely with the presence of activated glial cells than with the presence and amount of HIV-infected cells in the brain. Astrocytes, the most numerous cell type within the brain, provide an important reservoir for the generation of inflammatory mediators, including interferon-γ inducible peptide-10 (CXCL10), a neurotoxin and a chemoattractant, implicated in the pathophysiology of HAD. Additionally, the pro-inflammatory cytokines, IFN-γ and TNF-α, are also markedly increased in CNS tissues during HIV-1 infection. In the present study we hypothesized that the interplay of host cytokines and HIV-1 could lead to enhanced expression of the toxic chemokine, CXCL10. Our findings demonstrate a synergistic induction of CXCL10 mRNA and protein in human astrocytes exposed to HIV-1 and the pro-inflammatory cytokines. Signaling molecules, including JAK, STATs, MAPK (via activation of Erk1/2, AKT, and p38), and NF-κB were identified as instrumental in the synergistic induction of CXCL10. Understanding the mechanisms involved in HIV-1 and cytokine mediated up-regulation of CXCL10 could aid in the development of therapeutic modalities for HAD.
Astrocytes; HIV-associated dementia; CXCL10
Fc receptor-like 6 (FCRL6), the most recently characterized member of the FCRL family, is a cell surface glycoprotein with tyrosine-based regulatory potential. An extensive survey of human hematopoietic tissues disclosed that FCRL6 expression by NK and T cell subpopulations increases as a function of differentiation and is remarkably restricted to mature lymphocytes with cytotoxic capability. In particular, FCRL6 distinguishes perforin-expressing CD56dim NK cells, Vδ1+ and Vδ2+ γδ T cells, effector and effector memory CD8+ T cells, and rare cytotoxic CD4+ T cells in adult tissues. Analysis of this receptor in B cell chronic lymphocytic leukemia (CLL) was also performed. FCRL6 was found to mark significantly expanded populations of cytotoxic CD8+ T, CD4+ T, and NK cells in patients with CLL. Despite sequence homology with the known Fc receptors for IgG and IgE, FCRL6 did not bind immunoglobulin. Although FCRL6 can be tyrosine-phosphorylated, its antibody-mediated ligation was unable to influence cellular activation. Collectively these results demonstrate that FCRL6 is a distinct indicator of cytotoxic effector lymphocytes that is upregulated in diseases characterized by chronic immune stimulation.
Human NK cells; Human cytotoxic T cells; Fc Receptor-like; Chronic lymphocytic leukemia
To better understand how elephant seals (Mirounga angustirostris) use negative buoyancy to reduce energy metabolism and prolong dive duration, we modelled the energetic cost of transit and deep foraging dives in an elephant seal. A numerical integration technique was used to model the effects of swim speed, descent and ascent angles, and modes of locomotion (i.e. stroking and gliding) on diving metabolic rate, aerobic dive limit, vertical displacement (maximum dive depth) and horizontal displacement (maximum horizontal distance along a straight line between the beginning and end locations of the dive) for aerobic transit and foraging dives. Realistic values of the various parameters were taken from previous experimental data. Our results indicate that there is little energetic advantage to transit dives with gliding descent compared with horizontal swimming beneath the surface. Other factors such as feeding and predator avoidance may favour diving to depth during migration. Gliding descent showed variable energy savings for foraging dives. Deep mid-water foraging dives showed the greatest energy savings (approx. 18%) as a result of gliding during descent. In contrast, flat-bottom foraging dives with horizontal swimming at a depth of 400 m showed less of an energetic advantage with gliding descent, primarily because more of the dive involved stroking. Additional data are needed before the advantages of gliding descent can be fully understood for male and female elephant seals of different age and body composition. This type of data will require animal-borne instruments that can record the behaviour, three-dimensional movements and locomotory performance of free-ranging animals at depth.
diving; locomotion; gliding; aerobic dive limit; elephant seal
Induction of nitric-oxide synthase-2 (iNOS) by cytokines and bacterial products is associated with protein binding at the proximal promoter and in an upstream enhancer region of the Nos2 gene. To clarify how ethanol suppresses rat iNOS activity, we constructed several deletion mutants of the Nos2 promoter fused to the luciferase gene and transfected the constructs into C6 glial cells. Acute ethanol exposure of stably transfected cells for 24 h inhibits induced activity of Nos2 promoter constructs containing deletions in the 5′ flanking region, including a 94 bp promoter that lacks any known NF-κB site but which carries a C/EBPβ and overlapping γ-IRE, GAS and Oct motifs. Ethanol failed to inhibit the endogenous activity of a smaller, 78 bp promoter that lacks the C/EBPβ and overlapping, γ-IRE and GAS motifs and showed no inducible activity. As another approach, in vivo DNA footprinting was used and identified protein protections at five regions of the proximal Nos2 promoter in induced cells. Exposure to acute ethanol diminished protein occupation in the five promoter regions including the γ-IRE/NF-κB and the overlapping γIRE/GAS/Oct sites. Site-directed mutagenesis in the octamer domain of the γIRE/GAS/Oct motifs was studied in a 1002 bp promoter to examine its role in ethanol inhibition of cytokine and lipopolysaccharide induced activity. The data indicate ethanol failed to inhibit promoter activity when the Oct motif is missing. Electrophoretic mobility shift assays performed using a 22-mer probe containing the overlapping γ-IRE/GAS/Oct sites showed three complexes with one of the complexes being competed by an octamer-1 antibody. These observations demonstrate the role of protein-DNA binding at the core promoter, and the likely involvement of the octamer motif, in ethanol modulation of cytokine and lipopolysaccharide induced iNOS expression.
ethanol; Nos2; octamer; astrocyte; promoter
Emerging evidence indicates that neuroinflammatory responses in astroglia, including chemokine expression, are altered by opioids. Astroglial chemokines, such as CXCL10, are instrumental in response to many neuropathological insults. Opioid mediated disruption of astroglial CXCL10 expression may be detrimental in opioid abusers or patients receiving acute opioid therapy. We have characterized the in vitro effects of opioids on CXCL10 protein expression in human astroglial (A172) cells. The proinflammatory cytokine, tumor necrosis factor (TNF)α induced CXCL10 expression in A172 cells. Using MG-132, helenalin and SN50 [inhibitors of the transcription factor, nuclear factor (NF)-κB], we determined that NF-κB activation is instrumental in TNFα induced CXCL10 expression in A172 astroglia. Morphine exposure during the 24 h TNFα stimulation period did not alter CXCL10 expression. However, fentanyl, a more potent mu opioid receptor (MOR) agonist, inhibited TNFα induced CXCL10 expression. Interestingly, neither the nonselective opioid receptor antagonist, naltrexone nor β-funaltrexamine (β-FNA), a highly selective MOR antagonist, blocked fentanyl mediated inhibition of TNFα induced CXCL10 expression. Rather, β-FNA dose dependently inhibited TNFα induced CXCL10 expression with a greater potency than that observed for fentanyl. Immunoblot analysis indicated that morphine, fentanyl and β-FNA each reduced TNFα induced nuclear translocation of NF-κB p65. These data show that β-FNA and fentanyl inhibit TNFα induced CXCL10 expression via a MOR independent mechanism. Data also suggest that inhibition of TNFα induced CXCL10 expression by fentanyl and β-FNA is not directly related to a reduction in NF-κB p65 nuclear translocation. Further investigation is necessary in order to fully elucidate the mechanism through which these two opioid compounds inhibit CXCL10 expression. Understanding the mechanism by which chemokine expression is suppressed, particularly by the opioid antagonist, β-FNA, may provide insights into the development of safe and effective treatments for neuroinflammation.
astrocyte; brain injury; mu opioid receptor; neuroinflammation; tumor necrosis factor α
The FcRH4 transmembrane molecule, a member of the Fc receptor homologue family, can potently inhibit B cell receptor (BCR) signaling. We show that cell surface expression of this immunoregulatory molecule is restricted to a subpopulation of memory B cells, most of which lack the classical CD27 marker for memory B cells in humans. The FcRH4+ and FcRH4− memory B cells have undergone comparable levels of immunoglobulin isotype switching and somatic hypermutation, while neither subpopulation expresses the transcription factors involved in plasma cell differentiation. The FcRH4+ memory cells are morphologically distinctive large lymphocytes that express the CD69, CD80, and CD86 cell activation markers. They are also shown to be poised to secrete high levels of immunoglobulins in response to stimulation with T cell cytokines, but they fail to proliferate in response either to BCR ligation or Staphylococcus aureus stimulation. A heightened expression of the CCR1 and CCR5 chemokine receptors may facilitate their preferential localization in lymphoid tissues near epithelial surfaces. Cell surface FcRH4 expression thus marks a unique population of memory B cells with distinctive morphology, functional capabilities, and tissue localization.
Determination of reliable bioindicators of diabetes-induced oxidative
stress and the role of dietary vitamin E supplementation
were investigated. Blood (plasma) chemistries, lipid peroxidation
(LPO), and antioxidant enzyme activities were measured over
12 weeks in New Zealand White rabbits (control, diabetic, and diabetic +
vitamin E). Cholesterol and triglyceride levels did not correlate
with diabetic state. PlasmaLPOwas influenced by diabetes and
positively correlated with glucose concentration only, not cholesterol
or triglycerides. Liver glutathione peroxidase (GPX) activity
negatively correlated with glucose and triglyceride levels. Plasma
and erythrocyte GPX activities positively correlated with glucose,
cholesterol, and triglyceride concentrations. Liver superoxide dismutase
activity positively correlated with glucose and cholesterol
concentration. Vitamin E reduced plasma LPO, but did not affect
the diabetic state. Thus, plasmaLPOwas the most reliable indicator
of diabetes-induced oxidative stress. Antioxidant enzyme activities
and types of reactive oxygen species generated were tissue dependent.
Diabetes-induced oxidative stress is diminished by vitamin E